The NM_000203.5:c.300-3C>G variant in IDUA occurs within the splice acceptor region of intron 2. RT-PCR of fibroblast RNA from an individual who is compound heterozygous for the variant (labeled as 388-3C>G in the publication) revealed that the variant results in insertion of 58 intronic nucleotides, which would result in a frameshift). Analysis of heterozygous polymorphic markers showed that the variant caused a significant decrease in the level of IDUA mRNA derived from the allele with the c.300-3C>G variant. Furthermore, expression of the variant in COS-7 cells resulted in 1.6% wild type IDUA activity with no IDUA protein detected on western blot (PMID: 10735634) (PVS1). Two patients with this variant had documented IDUA deficiency within the affected range in leukocytes, urinary GAGs above the normal range, or clinical features specific to MPS I including hepatosplenomegaly, corneal involvement, skeletal and cardiac abnormalities (PMID: 10735634, 29801497). (PP4_Moderate). These individuals are compound heterozygous for the variant and another variant in IDUA, c.1037T>G or c.1874A>C) (PMID: 10735634, 29801497). The allelic data from these patients will be used in the classification of the second variant and is not included here to avoid circular logic (PM3 not applied). The highest population minor allele frequency in gnomAD v4.1.0 is 0.00004557 (2/44876 alleles) in the EAS population, which is lower than the ClinGen Lysosomal Diseases VCEP’s threshold for PM2_Supporting (<0.00025), meeting this criterion (PM2_Supporting). In summary, this variant meets the criteria to be classified as Pathogenic for MPS I based on the IDUA-specific ACMG/AMP criteria applied, as specified by the ClinGen Lysosomal Diseases Variant Curation Expert panel (Specifications Version 1.0.0): PVS1, PP4_Moderate, PM2_Supporting. (Classification approved by the ClinGen Lysosomal Diseases Variant Curation Expert Panel on May 23, 2025)